Podocyte protease activated receptor 1 stimulation in mice produces focal segmental glomerulosclerosis mirroring human disease signaling events
Authors: Carl J. May, Musleeha Chesor, Sarah E. Hunter, Bryony Hayes, Rachel Barr, Tim Roberts, Fern A. Barrington, Louise Farmer, Lan Ni, Maisie Jackson, Heidi Snethen, Nadia Tavakolidakhrabadi, Max Goldstone, Rodney Gilbert, Matt Beesley, Rachel Lennon, Rebecca Foster, Richard Coward, Gavin I. Welsh, Moin A. Saleem
Journal: Kidney International (2023)

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/−) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6. About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/−) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6. Translational StatementThere is compelling evidence implicating the role of a circulating factor in nephrotic syndrome, and we have previously shown that this factor signals via protease-activated receptor 1 (PAR-1). This work further extends our previous data showing that the signaling responses to PAR-1 agonists were conserved between 3 systems: podocytes in vitro, the mouse model expressing a transgenic overactive form of the PAR-1 receptor, and human circulating factor disease, demonstrating the significance. Looking downstream, TRPC6 (transient receptor potential cation channel subfamily c member 6) knockout podocytes demonstrated altered PAR-1 agonist signaling responses. Knockout of TRPC6 in the PAR-1–active mice significantly reduced their proteinuria and extended their lifespan. This work suggests that PAR-1 could be a key target in the treatment of nephrotic syndrome. There is compelling evidence implicating the role of a circulating factor in nephrotic syndrome, and we have previously shown that this factor signals via protease-activated receptor 1 (PAR-1). This work further extends our previous data showing that the signaling responses to PAR-1 agonists were conserved between 3 systems: podocytes in vitro, the mouse model expressing a transgenic overactive form of the PAR-1 receptor, and human circulating factor disease, demonstrating the significance. Looking downstream, TRPC6 (transient receptor potential cation channel subfamily c member 6) knockout podocytes demonstrated altered PAR-1 agonist signaling responses. Knockout of TRPC6 in the PAR-1–active mice significantly reduced their proteinuria and extended their lifespan. This work suggests that PAR-1 could be a key target in the treatment of nephrotic syndrome. Idiopathic nephrotic syndrome (INS) is one of the most difficult clinical conditions to manage in nephrology. Breakdown of the glomerular filtration barrier results in massive proteinuria, with the underlying trigger not well understood. A proportion of patients will not respond to any therapy, leading inevitably to renal failure and transplantation. In patients without a detectable genetic mutation, up to 50% suffer recurrence of disease.1Bierzynska A. Saleem M.A. Deriving and understanding the risk of post-transplant recurrence of nephrotic syndrome in the light of current molecular and genetic advances.Pediatr Nephrol. 2018; 33: 2027-2035Crossref PubMed Scopus (27) Google Scholar This leads to the reduced lifespan of the graft, and often after graft loss, the patient is deemed untransplantable given the very high risk of further recurrences. There is ample evidence that recurrent INS is caused by an as yet unknown circulating factor, likely produced by the immune system. Despite decades of study, the identity of such circulating factor(s) remains unknown.1Bierzynska A. Saleem M.A. Deriving and understanding the risk of post-transplant recurrence of nephrotic syndrome in the light of current molecular and genetic advances.Pediatr Nephrol. 2018; 33: 2027-2035Crossref PubMed Scopus (27) Google Scholar Here we have tested the hypothesis that the circulating factor works through podocyte protease-activated receptor 1 (PAR-1). PARs 1 to 4 are part of the well-known family of G protein–coupled receptors and are activated by serine proteases. PAR-1 and PAR-2 have been demonstrated to be expressed in the glomerulus, and immunohistochemical staining shows high PAR-1 expression in the podocytes.2Palygin O. Ilatovskaya D.V. Staruschenko A. Protease-activated receptors in kidney disease progression.Am J Physiol Renal Physiol. 2016; 311: F1140-F1144Crossref PubMed Scopus (35) Google Scholar Circulating plasma carries many proteases with diverse biological roles, which are tightly regulated by cognate antiproteases. We previously showed data suggesting that there is excess circulating protease activity in the plasma of post-transplant INS patients with active post-transplant disease (referred to as relapse plasma) that can signal to podocytes resulting in vasodilator-stimulated phosphoprotein (VASP) phosphorylation and increased podocyte motility, with the signaling being podocyte specific.3Harris J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar This effect is not present in response to remission plasma from the same patients or control plasma. Small, interfering RNA knockdown of PARs in podocytes demonstrated a role for the PAR-1 protease receptor in this response to relapse plasma. We hypothesized that there is a protease(s) that is present in the circulation that becomes dysregulated early in the pathogenesis of INS (either by upregulation or loss of a cognate antiprotease). Both T cells and memory B cells have been postulated as sources of the circulating factor,4Bierzynska A. McCarthy H.J. Soderquest K. et al.Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management.Kidney Int. 2017; 91: 937-947Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,5Oleinika K. Mauri C. Salama A.D. Effector and regulatory B cells in immune-mediated kidney disease.Nat Rev Nephrol. 2019; 15: 11-26Crossref PubMed Scopus (76) Google Scholar and our subsequent work suggests that the T helper cell 17 (Th17) subset could be a source of circulating proteases.6May C.J. Welsh G.I. Chesor M. et al.Human Th17 cells produce a soluble mediator that increases podocyte motility via signalling pathways which mimic PAR-1 activation.Am J Physiol Renal Physiol. 2019; 317: F913-F921Crossref PubMed Scopus (12) Google Scholar This increased activity of the circulating protease stimulates PAR-1 and induces specific signaling cascades. To test this at the receptor level, we now activated PAR-1 in vitro in conditionally immortalized human podocytes. We then used this signaling data to assess pathologic signaling in human nephrotic patient kidney biopsies and NPHS2 Cre PAR-1Active+/− mice. Furthermore, we hypothesized that podocyte TRPC6 (transient receptor potential cation channel subfamily c member 6) mediates PAR-1 signaling and is activated in response to human plasma or PAR-1 agonism. TRPC6 is an ion channel that binds to the podocyte-specific membrane protein podocin,7Dryer S.E. Reiser J. TRPC6 channels and their binding partners in podocytes: role in glomerular filtration and pathophysiology.Am J Physiol Renal Physiol. 2010; 299: F689-F701Crossref PubMed Scopus (122) Google Scholar and activating mutations in this channel in patients are able to cause focal segmental glomerular sclerosis (FSGS).8Hall G. Wang L. Spurney R.F. TRPC channels in proteinuric kidney diseases.Cells. 2019; 9: 44Crossref PubMed Google Scholar Both gain-of-function and loss-of-function mutations are known to cause FSGS.9Riehle M. Büscher A.K. Gohlke B.O. et al.TRPC6 G757D loss-of-function mutation associates with FSGS.J Am Soc Nephrol. 2016; 27: 2771-2783Crossref PubMed Scopus (79) Google Scholar In addition, it has been shown in podocytes that thrombin treatment can increase intracellular calcium concentrations. This increase is blocked by treatment with a TRPC3/6 inhibitor.10Guan Y. Nakano D. Zhang Y. et al.A protease-activated receptor-1 antagonist protects against podocyte injury in a mouse model of nephropathy.J Pharmacol Sci. 2017; 135: 81-88Crossref Scopus (19) Google Scholar The PAR-1 active construct was a kind gift of Dr. Shaun Coughlin, University of California San Francisco.11Hammes S.R. Shapiro M.J. Coughlin S.R. Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.Biochemistry. 1999; 38: 9308-9316Crossref PubMed Scopus (39) Google Scholar Briefly, all serine and threonine residues in the C-terminal tails have been substituted for alanines. This renders the PAR-1 receptor phosphor-null at the C-terminal tail. This form of the receptor is defective in both shutoff and agonist-triggered internalization. All animal experiments and procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986, and the Guide for the Care and Use of Laboratory Animals was followed during experiments. SV129 transgenic mice were generated by Genoway. These mice were bred with NPHS2 Cre hemizygous mice or NPHS2 rtTA Tet O Cre mice to generate developmental and inducible animals, respectively. The Pod Cre PAR-1 mice were crossed with whole-body TRPC6 knockout (KO) mice on a C57/Bl6 background. These mice were back-crossed to enrich for SV129. Inducible transgenic mice were fed 2 mg/ml doxycycline in 5% sucrose in their drinking water to activate the inducible transgene. Treatment started between 4 and 6 weeks of age and continued for 3 weeks. The start of doxycycline treatment has been designated week 0. The animals were culled at 3, 10, and 12 weeks, and their kidneys were examined. There is sufficient detail included in this paper to comply with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for the reporting of animal data. Human wild-type (WT) podocytes were conditionally immortalized using the SV40T antigen system. This allows podocytes to proliferate at the permissive temperature of 33 °C and differentiate at 37 °C.12Gallon L. Leventhal J. Skaro A. et al.Resolution of recurrent focal segmental glomerulosclerosis after retransplantation.N Engl J Med. 2012; 366: 1648-1649Crossref PubMed Scopus (33) Google Scholar Podocytes were cultured as previously described.13Ni L. Saleem M. Mathieson P.W. Podocyte culture: tricks of the trade.Nephrology (Carlton). 2012; 17: 525-531Crossref PubMed Scopus (66) Google Scholar Mouse podocyte cell lines were generated from a TRPC6 KO mouse on a C57 Bl/6 background as per the protocol detailed in a previous publication.13Ni L. Saleem M. Mathieson P.W. Podocyte culture: tricks of the trade.Nephrology (Carlton). 2012; 17: 525-531Crossref PubMed Scopus (66) Google Scholar Human glomerular endothelial cells were generated in-house and were conditionally immortalized using the same SV40T system as used in the podocytes. Mouse cell lines were generated by isolating glomeruli ex vivo, as described previously.14Varghese F. Bukhari A.B. Malhotra R. De A. IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples.PLoS One. 2014; 9e96801Crossref Scopus (778) Google Scholar Cultured podocytes were treated with PAR-1 agonist (Peptides International PAR-3665-PI) at a concentration of 15 μM for the indicated times. Podocytes were also treated with thrombin as a PAR-1 agonist (Sigma; #T6884) at a dose of 15 units/μl again for the indicated times. Podocytes were also treated with nephrotic patient plasma as previously described.3Harris J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar Each plasma pair was obtained from the same patient. Total protein lysates were extracted using a sodium dodecylsulfate lysis buffer supplemented with phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044; Sigma Aldrich). Blots were probed with primary antibodies: phospho–c-Jun N-terminal kinase (pJNK) Thr183/Tyr185 (#9251; Cell Signaling Technology), P p38 mitogen-activated protein kinase (MAPK) Thr180/Tyr182 (#9211; Cell Signaling Technology), β-actin (#A4700; Sigma), Phospho Paxillin Ser 178 (ab 193677; Abcam). All primaries were used following a 1:1000 dilution in 3% bovine serum albumin and incubated with the blot at 4 °C overnight. Primary antibodies were probed using a horseradish peroxidase–conjugated species-specific secondary antibody: either rabbit (#A0545; Sigma) or mouse (#A9044; Sigma). Secondaries were used following a 1:10,000 dilution in 3% bovine serum albumin and incubated with the blot for 1 hour at room temperature. The membranes were incubated with a synthetic analog of peroxide and with a signal enhancer, which are mixed in a 1:1 ratio (Geneflow; K1-0070). Blots were imaged using the Amersham Imager 600 from GE Healthcare Life Sciences. The densitometry in this study has been calculated using ImageJ. All densitometry represents the optical density of the band. This value is normalized to both a counterpart-actin load control and the mean of the untreated control bands of the antibody in question. Podocytes were cultured in 6-well plates. Scratch assays were performed once the podocytes had spent 14 days at the nonpermissive temperature of 37 °C. In brief, the media were aspirated, and a mechanical wound was inflicted by scratching the monolayer with a pipette tip. Podocytes were then washed in phosphate-buffered saline twice to remove any debris and promigratory factors. The wells were then treated, and the scratch area was imaged after 0 (control) and 12 hours using a Leica DMIRB microscope and a Zeiss Axiocam Erc 5S camera. The area of the clear zone was measured over time, and podocyte migration was assessed by reduction in the area, indicating more motile cells. PAR-1Active+/− mice were generated by Genoway, France. The NPHS2 Cre and NPHS2 Cre rtTA mice were a gift from Professor Susan Quaggin. TRPC6 KO mice were purchased from the Jackson Laboratory. Kidneys were harvested and immersed in formalin before being paraffin embedded. Kidneys were sectioned by our in-house histology facility. Masson's trichrome stain was performed using a kit (HT15-1KT; Sigma), and periodic acid–Schiff stain was also performed using a kit (395B; Sigma). The first 6 to 8 glomeruli alighted upon during looking at the slide were imaged. The glomeruli were manually isolated from the rest of the image. The immunohistochemistry (IHC) was quantified using an open-source plugin for ImageJ called the "IHC profiler." Therefore, the IHC profiler measures the glomerular signal only. The plugin is described in detail here.15Foster R.R. Slater S.C. Seckley J. et al.Vascular endothelial growth factor-C, a potential paracrine regulator of glomerular permeability, increases glomerular endothelial cell monolayer integrity and intracellular calcium.Am J Pathol. 2008; 173: 938-948Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar A pathologist (MB) was sent 48 images of the glomeruli from the developmental mice, 24 from each of the control and PAR-1Active+/− mice. MB was blinded to the genotype associated with each image. Images were scored for glomerulosclerosis. A more detailed description of this assay can be found here.16Yao F. He Z. Lu M. et al.Smad2 and Smad3 play differential roles in the regulation of matrix deposition-related enzymes in renal mesangial cells.Int J Clin Exp Med. 2017; 10: 10161-10169Google Scholar Briefly, the Rnorm was plotted against time, and the peak to baseline ratios of Rnorm were calculated as a measure of the maximal effect on [Ca2+]i. Graphs and statistical analyses were compiled and performed using GraphPad Prism (GraphPad Software). The following asterisks have been used to denote statistical significance: ∗P ≤ 0.05, ∗∗P ≤ 0.01, ∗∗∗P ≤ 0.001, ∗∗∗∗P ≤ 0.0001. Biopsy tissue was obtained via the UK Nephrotic Syndrome Study, NephroS, housed within the UK Renal Rare Disease Registry, RaDaR. The PAR-1 receptor is highly expressed by the podocyte, as shown in the Sigma protein atlas17Kellenberger T. Krag S. Danielsen C.C. et al.Differential effects of Smad3 targeting in a murine model of chronic kidney disease.Physiol Rep. 2013; 1e00181Crossref PubMed Scopus (14) Google Scholar (Figure 1a), strikingly in Nephrocell RNA expression data18Humphreys Lab. Kidney regenerative medicine.http://humphreyslab.com/Date accessed: June 1, 2021Google Scholar and in single-cell RNA databases.19López-Colomé A.M. Lee-Rivera I. Benavides-Hidalgo R. Paxillin López E. a crossroad in pathological cell migration.J Hematol Oncol. 2017; 10: 50Crossref PubMed Scopus (201) Google Scholar Conditionally immortalized human podocytes were treated with PAR-1 agonist (Figure 1b–e). This agonist is a short peptide and is highly specific for the PAR-1 receptor. This treatment stimulated a significant increase in the phosphorylation of VASP at serine 157 (Figure 1b). Previous work published by the group has shown a similar response to NS patient plasma, which was reduced after knockdown of the PAR-1 receptor.3Harris J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar In addition, we now show that p38 MAPK, JNK, and Paxillin all displayed significant increases in phosphorylation in response to PAR-1 agonist treatment (Figure 1c–e, respectively). The phospho-VASP (pVASP), JNK, and Paxillin phosphorylation events are podocyte specific; no such phosphorylation was seen in PAR-1 agonist–treated glomerular endothelial cells (Supplementary Figure S1). However, phosphorylation of p38 MAPK was observed showing that the glomerular endothelial cells are PAR-1 sensitive. Phosphorylation of paxillin at serine 178 is associated with an increase in cell motility.20Welsh G.I. Saleem M.A. The podocyte cytoskeleton—key to a functioning glomerulus in health and disease.Nat Rev Nephrol. 2011; 8: 14-21Crossref PubMed Scopus (199) Google Scholar Several other signaling pathways were interrogated but revealed no significant stimulation in response to PAR-1 agonist (Figure 1f). Wound-healing assays revealed that PAR-1 agonist treatment significantly increased podocyte motility (Figure 1g), which is considered a surrogate of increased podocyte effacement in vivo.21Scheuble J. Rössler O.G. Ulrich M. Thiel G. Pharmacological and genetic inhibition of TRPC6-induced gene transcription.Eur J Pharmacol. 2020; 886173357Crossref PubMed Scopus (4) Google Scholar PAR-1 activating peptide treatments were repeated along with a TRPC6 inhibitor (SAR 7334 Tocris).22Foster R.R. Zadeh M.A. Welsh G.I. et al.Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells.Cell Calcium. 2009; 45: 384-390Crossref PubMed Scopus (33) Google Scholar Stimulation with PAR-1 agonist significantly stimulated the p38 MAPK (Figure 2a), JNK (Figure 2b), and VASP (Figure 2c) pathways. The inhibitor significantly reduced the signaling response to PAR-1 agonist treatment for p38 MAPK, JNK, and VASP. Thrombin (27 μM) significantly stimulated the p38 MAPK, JNK, and VASP signaling pathways (Each Bonferroni's Multiple Comparison Test). SAR7334 (10 nM with 30-minute preincubation) treatment significantly reduced p38 MAPK, JNK, and VASP stimulation. Patient relapse plasma also significantly stimulated the p38 MAPK, JNK, and VASP pathways compared with remission plasma treatment (Figure 2d–f). This signaling response could be significantly reduced using the TRPC6 inhibitor SAR7334. Biopsies from patients with NS were obtained via the UK Renal Rare Disease Registry, RaDaR (Supplementary Figure S2). The biopsies were stained for pVASP, pJNK, and pPaxillin. Significant increases in the glomerular phosphorylation of VASP were seen in patients with minimal change disease and FSGS (pretransplant) compared with those with a noncirculating factor disease, IgA nephropathy (Figure 3a–c). Patient 643 has Frasier syndrome, which is a genetic condition caused by a WT1 mutation that causes FSGS. Interestingly, there was no pVASP signal in these biopsies. A significant increase in pJNK was seen in patients with FSGS versus patients with IgA. Again patient 643 exhibited no pJNK signal (Figure 3d–f). Overactivation of PAR-1 in vivo, exclusively in the podocyte, caused glomerular damage with histologic features that closely resemble human FSGS pathogenesis. A construct encoding a C-terminal phospho-null form of the PAR-1 receptor was cloned into the ROSA 26 locus of SV129 mice. This form of the PAR-1 receptor is constitutively active because it remains at the membrane once activated rather than being rapidly shuttled to the lysosome for degradation. We initially confirmed the specific location of PAR-1 in the podocyte by detecting the FLAG-tag protein specifically in these cells (Figure 4a). The NPHS2 Cre PAR-1Active+/− (developmental) mice expressed transgenic PAR-1 following cleavage by Cre recombinase (Supplementary Figure S3), were born at normal frequency, and were indistinguishable from their control littermates initially. Postnatally, the NPHS2 Cre PAR-1Active+/− mice became progressively more albuminuric. By 32 days of age, they were grossly albuminuric (Figure 4b). NPHS2 Cre PAR-1Active+/− mice were also in renal failure as evidenced by their significantly higher levels of serum creatinine and urea compared with littermate controls (Figure 4c and d, respectively). Furthermore, NPHS2 Cre PAR-1Active+/− mice died between the ages of 39 and 45 days (Figure 4e). Littermate controls were healthy with normal lifespans. Mice were culled at days 8, 21, 32, and 40. The day 8 NPHS2 Cre PAR-1Active+/− mice had indistinguishable glomerular histology compared with controls (Figure 4f). By day 32 on both the periodic acid–Schiff and trichrome images, there was an accumulation of matrix within the glomerulus, and this matrix is largely composed of collagen. At day 32, the glomeruli of these animals were clearly fibrotic. By 40 days of age (where death from kidney failure in the mutants is imminent), there were clear signs of glomerulosclerosis. This progression of histologic features is reminiscent of human FSGS. Electron microscopy studies demonstrated that at day 1 of age, the kidneys in the mutant mice appeared ultrastructurally normal. However, as early as day 26, the fine ultrastructure of the glomerular basement membrane was becoming deranged, with complete effacement of podocyte foot processes (Figure 4g). It was found that only the PAR-1Active+/− mice showed evidence of sclerosis, whereas the control animals showed no evidence of sclerosis (Figure 4h and i). There was a significant increase in sclerosis in the PAR-1Active+/− mice when looked at by individual mouse and a significant increase in sclerosis when the mice were collated by genotype. A podocyte-specific inducible PAR-1Active+/− mouse line was generated to investigate to what extent activation of podocyte PAR-1 in adult mice reproduces this phenotype. The mutants were induced at between 6 and 8 weeks of age with 2 mg/ml doxycycline. The inducible mutants demonstrated clear signs of NS within 3 weeks including significantly increased levels of albuminuria and glomerular mesangial expansion on periodic acid–Schiff stain (Figure 5a and b ). The progression of albuminuria and histologic changes were similar to the results seen in the developmental model, including an initial hypercellularity followed by mesangial expansion with fibrosis and sclerosis. Blind scoring by a pathologist (MB) revealed a significant increase in segmental sclerosis in the inducible mutants (Figure 5c and d). By IHC, there was a significant increase in VASP phosphorylation in the glomeruli of the NPHS2 Cre PAR-1Active+/− mice at day 8 (Figure 6a). There were also significant increases in the staining of pJNK (Figure 6b), all seen in a podocyte distribution. pPaxillin signaling was also significantly increased in the glomeruli of the NPHS2 Cre PAR-1Active+/− mice at day 8 (Figure 6c). We noticed that 1 of 6 PAR-1Active +/− was a relative outlier for pJNK and pPaxillin, but the trend for all the rest was in the same direction. IHC was performed on kidney sections from 3 mice, with 6 to 8 glomeruli analyzed per mouse. This is the same signaling response as seen in the human podocytes in vitro in response to both PAR-1 agonist and relapse plasma. Flufenamic acid is a specific TRPC6 agonist that stimulates calcium influx in podocytes.23Farmer L.K. Rollason R. Whitcomb D.J. et al.TRPC6 binds to and activates calpain, independent of its channel activity, and regulates podocyte cytoskeleton, cell adhesion, and motility.J Am Soc Nephrol. 2019; 30: 1910-1924Crossref PubMed Scopus (45) Google Scholar This calcium influx was potentiated by treatment with relapse plasma from nephrotic patients compared with remission plasma (Figure 7a). To test if TRPC6 mediates podocyte signaling pathways downstream of PAR-1, a TRPC6-null mouse podocyte cell line was treated with PAR-1 agonist peptide.24Wang L. Chang J.H. Buckley A.F. Spurney R.F. Knockout of TRPC6 promotes insulin resistance and exacerbates glomerular injury in Akita mice.Kidney Int. 2019; 95: 321-332Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar Significant phosphorylation of JNK and VASP was seen in WT mouse podocytes with no significant phosphorylation in TRPC6 KO podocytes (Figure 7b and c, respectively). Similarly, for paxillin, there seemed to be evidence of stimulation of the WT podocytes after PAR-1 agonist treatment with no evidence of stimulation of paxillin in the TRPC6 KO mice (Figure 7d). It is clear that the TRPC6 KO podocytes demonstrate a blunted promotility response to PAR-1 agonist treatment. In vivo, we crossed the Pod Cre PAR-1Active+/− mouse with a TRPC6−/− mouse to generate the developmental PAR-1Active mouse on a TRPC6-null background. The whole-body KO of TRPC6 can exacerbate glomerular disease but is not sufficient to cause disease.25Singh I. Knezevic N. Ahmmed G.U. et al.Galphaq-TRPC6-mediated Ca2+ entry induces RhoA activation and resultant endothelial cell shape change in response to thrombin.J Biol Chem. 2007; 282: 7833-7843Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar The TRPC6 KO mice crossed with PAR-1Active had significantly reduced proteinuria compared with PAR-1Active mice (Figure 7e). TRPC6 KO in the Pod Cre PAR-1Active+/− significantly improved lifespan (Figure 7f). These animals are on a mixed background including the C57 Bl6 strain, which are known to be resistant to proteinuria. Indeed, the control mice on this mixed background are less proteinuric than their counterparts on a singleSV129 background. Blind scoring by a pathologist (MB) revealed no significant change in glomerulosclerosis between the WT and TRPC6KO variants of the PAR-1–active mice (Figure 7g and h). There was a significant reduction in glomerular pVASP signaling in the mice at 8 days in the Pod Cre PAR-1Active+/− TRPC6KO mice versus the Pod Cre PAR-1Active+/− TRPC6 WT mice (Figure 8a). There was no significant difference between the 2 m